A long-term goal of the BRAIN Initiative is to track activity in large numbers of neurons individually in behaving animals, thus capturing the information processing that is done by brain circuitry. A universal signal in neurons when they are active is the messenger ion calcium. By examining changes in calcium concentration following each action potential in an individual neuron, researchers can track that neuron's activity, but at present, the time resolution of this approach is limited. The overall objective of this application is to use calcium-sensitive probes in spike-counting mode to detect individual action potentials as discrete events in space and time. The proposed experiments will produce new technology that can follow activity with millisecond resolution in hundreds to thousands of neurons at once. This contribution is significant because it will allow researchers to image important neural circuits noninvasively with millisecond resolution in awake animals. This approach is innovative because the investigators will use a novel, ultrafast response mode of a highly popular calcium sensor protein to obtain time resolution that is not otherwise possible. The work proposed in this application therefore advances our ability to understand how the brain functions in health and disease. In the long run, this information could lead to new approaches to diagnosis, treatment, and prevention of a variety of brain disorders.